Quantitation of free and total glycerin content in biodiesel is performed via a simple and direct ambient desorption/ionization mass spectrometric technique. Easy ambient sonic-spray ionization mass spectrometry (EASI-MS) is applied to either the crude or derivatized sample spiked with an internal standard allowing for immediate quantitation of free and total glycerin, a major quality parameter for biodiesel. The linear range obtained by EASI-MS for free glycerin was from 0.005 to 0.100% (m/m), and that for bound glycerin (mono-, di-, and triacylglycerides) was from 0.01 to 0.50% (m/m). These values are in concordance with those required by major regulatory agencies for biodiesel quality control, which are, for free glycerin, a 0.02% (m/m) maximum and, for total glycerin, a 0.25% (m/m) maximum. The simple and fast EASI(+)-MS protocol seems to offer a proper substitute or alternative for the more demanding and time-consuming gas chromatography with flame ionization detection (GC-FID) standard methodology.